Regulation of bone mass is a highly complex process involving the coordinated actions of osteoblasts (OBs), osteoclasts, and osteocytes. Aberrant signaling is manifest in a host of disease states such as osteoporosis, fracture, and cancer. This proposal will examine for the first time the direct role of alternative NF-?B in the OB lineage during basal bone homeostasis and under stress conditions. Transgenic mouse models will be used to modulate alternative NF-?B in the OB lineage using Osx-Cre. Upon induction of alternative NF-?B, NIK phosphorylates IKKa. As a NIKf/f model does not currently exist, Osx-Cre;IKKaf/f animals (cKO) will be used to examine inhibition of alternative NF-?B in the OB lineage. NIKdeltaT3 (NT3) is a constitutively active allele of NIK placed downstream of a floxed STOP cassette. Recombination with Osx-Cre will activate alternative NF-?B in the OB lineage (cACT). In Aim 1a, microCT, histomorphometric and mechanical strength analyses of control, cKO, and cACT mice will establish how loss or activation of the alternative NF-?B pathway in the OB lineage impacts basal bone phenotype. In Aim 1b, the in vitro OB phenotype of cKO and cACT cultures will be compared to control. Addition of known activators (Wnt3a, BMP2) of osteogenesis will delineate possible stage-specific effects of alternative NF-?B and interactions with various osteogenic signals. In Aim 2, the response to either pathological mechanical or hormonal anabolic stimuli will be evaluated in the same animals used in Aim 1. Aim 2a will use an in vivo loading protocol to monitor OB lineage cell response to mechanical stress. In Aim 2b, the ability of OB lineage cells to respond to hormonal stimulus will be evaluated by treating animals with intermittent parathyroid hormone, a currently used osteoporosis therapy. Bone formation rate and bone mass will be quantified in both Aims 2a and 2b to assess how loss or activation of the alternative NF-?B pathway influences the ability of OB lineage cells to respond to these anabolic stimuli. Thus, both in vivo and in vitro experiments will test the overarching hypothesis that alternative NF-?B is a negative regulator of OB lineage differentiation and/or function. The results of this proposal will provide valuable insight into the molecular mechanisms of osteogenesis, which may help guide future interventions to treat bone-related pathologies.